Heme is a red pigment comprised of four sub-units called pyrroles which are chemically united to form a single large tetrapyrrole (porphyrin) ring structure. A metal ion is chelated at the center of this porphyrin. In higher organisms this metal is iron and the resulting structure is called iron protoporphyrin.
In physiological systems, heme is bound to certain proteins and these heme proteins bind oxygen at the site of the iron atom or they function as components of membrane bound electron transport systems. Cellular respiration, energy generation and chemical oxidation are dependent on these heme proteins.
In mammals and other vertebrates, heme is catabolized by the enzyme heme oxygenase to produce the bile pigment, bilirubin. Biliverdin is reduced to bilirubin by the action of the enzyme biliverdin reductase. In liver, bilirubin is converted to mono- and di-glucoronide conjugates which are excreted to the bile. The spleen is responsible for about 75 percent of bilirubin production, the remainder being produced in other body organs such as the liver and the kidneys, with the liver being responsible for 15-25 percent production of the total bilirubin. Bilirubin is a neurotoxic compound, but this toxicity does not normally present a serious problem in adults since, as was previously stated, bilirubin is converted to the corresponding di-glucoronide. The di-glucoronide is water soluble and is excreted. One of the more difficult aspects of the toxicity of bilirubin is the so-called jaundice of the newborn resulting from high concentrations of bilirubin in the blood serum of newborn mammals. Free bilirubin is fat soluble and readily crosses the blood-brain barrier causing extensive and serious brain damage. One manifestation of such toxicity in the brain is kernicterus or bilirubin encepthalopathy.
Elevated levels of bilirubin often appear in the serum of individuals with diseases such as congenital anemia, thalassemia and sickle cell anemia as well as various forms of liver disease. The concentration of bilirubin in the serum of such individuals, however, rarely reaches the high level found in neonates, although it can attain toxic levels which should be controlled.
The problem of bilirubin toxicity in mammals has received the attention of numerous investigators. Several recent patents, for example, have addressed this problem and disclosed therapeutic compositions which are used to decrease the rate at which heme is metabolized in mammals. A few of these patents, which are assigned to the assignee of the invention described herein, are discussed below.
U.S. Pat. No. 4,657,902 relates to the compound tin mesoporphyrin (SnMP) and therapeutic compositions containing the same used to inhibit heme metabolism in mammals, control the rate of tryptophan metabolism and increase the rate at which heme is excreted.
U.S. Pat. Nos. 4,668,670 and 4,692,439 are both directed to tin diiododeuteroporphyrin (SnDDP) for similar purposes.
U.S. Pat. No. 4,684,637 discloses the use of tin protoporphyrin (SnPP) for decreasing the rate of heme metabolism in mammals.
While the compounds disclosed in the foregoing patents cause a reduction in the catabolic activity of heme oxygenase, resulting in decreased bilirubin production, they would do so more efficiently if their concentration in the spleen could be increased. The spleen is the major producer of bilirubin. It is desirable, therefore, to increase the amount of active metalloporphyrin which reaches the spleen.
Attempts have been made to target the porphyrin compounds to different sites or organs in mammals. For example, G. Jori et al in their article on "Controlled Targeting of Different Subcellar Sites by Porphyrins in Tumor-bearing Mice," Brit. J. Cancer, Vol. 53, pp. 615-621 (1986), disclose that intraperitoneal injection of liposome-bound porphyrins to mice results in more efficient tumor targeting than that obtained by administration of the same porphyrins dissolved in homogeneous aqueous solutions. These investigators were not concerned with selective targeting of normal body organs of tissue enzymes. Indeed, in a later article on "Utilization of Liposomes and Low Density Lipoproteins and Porphyrin Carriers in Experimental Photodynamic Therapy," First International Conference on the Clinical Applications of Photosensitization for Diagnosis and Treatment, Tokyo, Japan (1986), Jori discloses that the administration of liposome-vehiculated hematoporphyrin (Hp) to the tumor tissue and to the organs of the reticuloendothelial system of mice did not result in any specificity of Hp localization although it did result in larger accumulation of Hp in the tumor.
While others have studied and reported on the use of liposomes as carriers for various drugs, so far as it is known none of these investigations have succeeded in selectively targeting a specific body organ or enzyme contained therein, to increase the concentration at the site of action of the porphyrin compound after it is administered to mammals.
It has also been demonstrated that certain metalloporphyrins are effective in decreasing bilirubin levels in various mammals, including rats, monkeys and humans. Table I below shows the effect of SnPP, and where indicated, CrPP and ZnPP on reduction of bilirubin levels in various mammals.
TABLE I ______________________________________ BILIRUBIN/ SnPP DOSE CO REDUC- ANIMAL MODEL & SPECIES (umoles/kg) TION (percent) ______________________________________ BILIARY CIRRHOSIS-MAN 0.25-2.0 7-23 GILBERT'S DISEASE-MAN 0.5-1.8 29-43 NORMAL ADULT-MAN 1-2 38-47 ABO INCOMPATIBILITY- 0.5 21-32 MAN NEONATAL MONKEY 24-185 91 NORMAL ADULT RAT 10-50 24-30 BILE DUCT LIGATED RAT 100 62-70 NEONATAL RAT 10(CrPP) 32 NEONATAL RAT/ 65 25 HEMATOMA NEONATAL GUNN RAT 50 19 NORMAL ADULT RAT 4-40(ZnPP) 20-30 ______________________________________
The data in Table I indicate that these metalloporphyrins inhibit heme oxygenase in humans, rats and monkeys, and thus appreciably reduce the bilirubin and/or bilirubin and carbon monoxide production rates. Where dosage ranges and bilirubin reduction ranges are given in the table, they represent the ranges for several mammals studied.
It has also been observed that following intravenous administration of SnPP into rats, the highest concentrations of SnPP were found in the liver and kidneys while the concentration of SnPP in the spleen was one-fourth of SnPP concentration in the liver. In addition, although splenic heme oxygenase was inhibited by 50-75%, the heme oxygenase activity was still high. It was, in fact, about the same as the heme oxygenase activity found in the livers of the control animals. The results of these studies are shown in Table II.
TABLE II __________________________________________________________________________ ITEM KIDNEY LIVER SPLEEN __________________________________________________________________________ CONCENTRATION OF SnPP IN ORGAN @ 24 HOURS (A) HOMOGENATE (.mu.M) 99.3 .+-. 1/5* 27/0 .+-. 1.8 6.5 .+-. 0.6 (B) MICROSOMES -- 146-284 60 -90 (pm/mg. protein) HEME OXYGENASE ACTIVITY (nm bilirubin/hr/mg. protein) (A) PRIOR TO TREATMENT 1.8 .+-. 0.2 2.7 .+-. 0,3 12.4 .+-. 0.4 (B) 24 HOURS AFTER 0.6 .+-. 0.1 0.3 .+-. 0.04 2.3 .+-. 0.3 TREATMENT WITH AQUEOUS SnPP __________________________________________________________________________ *Mean .+-. SE for 3-4 rats
Despite all of the studies and efforts described above, the problem of targeting active porphyrins to the spleen where they would be most effective in binding and inhibiting the enzyme, heme oxygenase, remains unsolved.
Accordingly, it is an object of this invention to inhibit or substantially reduce the catabolic action of heme oxygenase in mammals.
It is another object of this invention to inhibit or substantially reduce the catabolic action of heme oxygenase in a selected body organ of mammals, notably humans.
It is a further object of this invention to inhibit or substantially reduce the catabolic action of heme oxygenase in a selected body organ by the administration of a porphyrin compound bound to liposome.
It is still another object of this invention to inhibit or substantially reduce the catabolic action of heme oxygenase in the spleen of mammals by selectively targeting the spleen with a liposome-porphyrin product administered to the mammals.
It is also an object of this invention to increase the concentration of heme oxygenase activity inhibiting porphyrins in the spleen of mammals by administering a liposome porphyrin product to the mammal and selectively targeting the heme oxygenase in the spleen.
The foregoing and other objects and features of the present invention will be more clearly comprehended from the following detailed description of the invention taken together with the accompanying drawings.